WO2018181997A1 - Procédé pour la détection d'une bactérie produisant de la carbapénémase - Google Patents

Procédé pour la détection d'une bactérie produisant de la carbapénémase Download PDF

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WO2018181997A1
WO2018181997A1 PCT/JP2018/013920 JP2018013920W WO2018181997A1 WO 2018181997 A1 WO2018181997 A1 WO 2018181997A1 JP 2018013920 W JP2018013920 W JP 2018013920W WO 2018181997 A1 WO2018181997 A1 WO 2018181997A1
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absorbance
abs
wavelength
carbapenemase
sample
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Japanese (ja)
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壇 竹内
茂幸 浜田
和典 朝野
幸宏 明田
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国立大学法人大阪大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention relates to a method for detecting a carbapenemase-producing bacterium, an apparatus used for the method, a method for determining the hydrolysis performance of a carbapenemase-producing bacterium, and a method for screening an inhibitor of a carbapenemase-producing bacterium.
  • Carbapenemase-producing Enterobacteriaceae decomposes carbapenem antibiotics, which are trump therapies for severe infections, and exhibits resistance, and its worldwide spread is a major problem. Therefore, establishing a CPE detection system has great clinical significance, and several reports have been reported so far.
  • Patent Document 1 relates to a disk diffusion method, and specifically, a disk test containing at least three kinds of antibacterial drugs consisting of a carbapenem antibacterial agent, a fluoroquinolone antibacterial agent and an aminoglycoside antibacterial agent.
  • a disk test containing at least three kinds of antibacterial drugs consisting of a carbapenem antibacterial agent, a fluoroquinolone antibacterial agent and an aminoglycoside antibacterial agent.
  • Patent Document 2 discloses that by using a composition in which faropenem and cloxacillin are combined in a micro liquid dilution method, only carbapenemase-producing resistant bacteria can be selectively grown, and carbapenemase-producing resistant bacteria can be detected easily and accurately. ing.
  • the conventional technique requires a complicated operation and time for the preparation of the measurement sample, and the evaluator's skill level is necessary for the judgment, so it can be carried out easily and quickly with high sensitivity and specificity. Establishment of a new system was required.
  • An object of the present invention is to provide a method for detecting a carbapenemase-producing bacterium, a device for detecting a carbapenemase-producing bacterium, a method for determining the hydrolysis performance of the carbapenemase-producing bacterium, and a carbapenemase-producing bacterium that can be carried out simply and quickly with high sensitivity and specificity. It is related with providing the method of screening the inhibitor.
  • the present invention relates to the following [1] to [9].
  • a method for detecting a carbapenemase-producing bacterium comprising the following steps. Step 1: Step of culturing by adding an antibacterial agent to a sample Step 2: Absorbance (A-Abs) at the maximum wavelength (Anm) of the antibacterial agent with respect to the culture solution obtained in Step 1, and the wavelength Step 3 for measuring absorbance (B-Abs) at a wavelength (Bnm) of at least 40 nm higher wavelength from step 3: Applying the absorbance (B-Abs) obtained in step 2 to a calibration curve, and calculating the blank absorbance (C-Abs) Calculation step Step 4: When the difference between the absorbance (A-Abs) obtained in Step 2 and the absorbance (C-Abs) obtained in Step 3 is below a preset threshold, the sample contains a carbapenemase-producing bacterium [2] The detection method according to [1], wherein the wavelength (Bnm) is 45 to 400 n
  • [3] The detection method according to [1] or [2], wherein imipenem, panipenem, meropenem, biapenem, doripenem, tevipenem, ertapenem, or faropenem is used as the antibacterial agent.
  • [4] The detection method according to any one of the above [1] to [3], wherein the sample is a biological specimen derived from a human or other animal, an environmental specimen or a food specimen.
  • [5] The detection method according to any one of [1] to [4] above, wherein the culture time in step 1 is at least 15 minutes.
  • a spectrophotometric measurement apparatus, and a computer including a processor and a memory under the control of the processor, the memory including the following steps: Absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent and a wavelength at least 40 nm higher than the wavelength of the culture solution cultured with the sample and the antibacterial agent added thereto.
  • A-Abs Absorbance
  • Step 1 Step of culturing by adding an antibacterial agent to a sample
  • Step 2 Absorbance (A-Abs) at the maximum wavelength (Anm) of the antibacterial agent with respect to the culture solution obtained in Step 1, and the wavelength Step 3 for measuring absorbance (B-Abs) at a wavelength (Bnm) of at least 40 nm higher wavelength from step 3: Applying the absorbance (B-Abs) obtained in step 2 to a calibration curve, and calculating the blank absorbance (C-Abs) Calculation step 4: When the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold, the carbapenemase-producing bacterium contained in the sample [9] The absorbance (A-Abs) at the wavelength (Anm) of the sample obtained by allowing the test substance to act on the carbapenemase-producing bacterium, and the wavelength (Bnm) at least 40 nm higher than the wavelength
  • the test substance is selected as a substance that suppresses the activity of the carbapenemase-producing bacteria.
  • a carbapenemase-producing bacterium can be detected simply and quickly with high sensitivity and specificity.
  • FIG. 1 shows the results of measuring the absorbance spectrum of each antibacterial drug
  • FIG. 1a shows the measurement results at wavelengths of 220 to 400 nm
  • FIG. 1b shows the correlation between the concentration of imipenem and the absorbance at 297 nm
  • FIG. 2 shows the result of measuring the spectrum of each antibacterial agent when bacteria are present
  • FIG. The measurement results at 400 nm are shown in Fig. 2b, in which the spectrum in the case where the antibacterial agent and the bacterium coexist is compared with the spectrum in the case where the bacterium is present in the PBS for each antibacterial agent.
  • FIG. 3 is a diagram showing the absolute absorbance of imipenem in the bacterial mixture.
  • FIG. 3A schematically shows the absolute absorbance from the absorbance spectra of the bacterial mixture containing imipenem and the bacterial mixture not containing imipenem.
  • FIG. 3b is a diagram showing the absolute absorbance spectrum by subtracting the absorbance of the bacterial mixture not containing imipenem.
  • FIG. 4 is a diagram schematically showing a solution prepared for absorbance measurement.
  • FIG. 4a is a case where a solution containing bacteria and imipenem (experimental solution) and a solution containing only bacteria at the same concentration (background) are prepared.
  • FIG. 4 b shows a case where the preparation of the background solution is omitted.
  • FIG. 5 is a graph showing the correlation between the background absorbance at 297 nm (C-Abs) and the absorbance at a wavelength different from 297 nm (B-Abs), and FIG. 5a shows the absorbance at the wavelength of 350 nm (B-Abs).
  • 5b is a graph showing the correlation when the absorbance (B-Abs) at a wavelength of 450 nm is used
  • FIG. 5c is a graph showing the correlation when the absorbance (B-Abs) at a wavelength of 600 nm is used.
  • FIG. 6 is a graph showing the hydrolysis rate of imipenem.
  • FIG. 6a shows the result of comparing the hydrolysis rate between CPE and non-CPE strains (55 strains), and FIG.
  • FIG. 6b shows the hydrolysis rate in the CPE strain. It is a figure which shows the level of performance.
  • FIG. 7 is a diagram showing the results of comparing hydrolysis rates between CPE strains and non-CPE strains using antibacterial agents.
  • FIG. 8 is a diagram showing the hydrolysis rate of imipenem.
  • FIG. 8a shows the result of comparing the hydrolysis rate between CPE and non-CPE strains (149 strains), and
  • FIG. 8b shows the hydrolysis in the CPE strain. It is a figure which shows the level of performance.
  • FIG. 9 is a diagram showing the results of comparing the hydrolysis rate between the CPE strain and the non-CPE strain for each incubation time.
  • the present invention utilizes the hydrolysis reaction of an antibacterial agent by a carbapenemase-producing bacterium, and is based on the fact that the specific ultraviolet region absorption of the antibacterial agent changes due to hydrolysis.
  • the present invention has been found by focusing on the fact that the anti-bacterial drug hydrolyzed by carbapenemase changes the ultraviolet absorption spectrum of the anti-bacterial drug with hydrolysis. That is, when measuring the absorbance of the antibacterial drug, a wavelength at which the absorbance varies due to hydrolysis and a specific wavelength different from the wavelength as the background are set, and the absorbance at these wavelengths is measured from the same sample.
  • detecting a carbapenemase-producing bacterium refers to detecting whether or not a carbapenemase-producing bacterium is contained in a sample and determining whether or not a carbapenemase-producing bacterium is present.
  • Step 1 Step of culturing by adding an antibacterial agent to a sample
  • Step 2 Absorbance (A-Abs) at the maximum wavelength (Anm) of the antibacterial agent with respect to the culture solution obtained in Step 1, and the wavelength Step 3 for measuring absorbance (B-Abs) at a wavelength (Bnm) of at least 40 nm higher wavelength from step 3: Applying the absorbance (B-Abs) obtained in step 2 to a calibration curve, and calculating the blank absorbance (C-Abs) Calculation step
  • Step 4 When the difference between the absorbance (A-Abs) obtained in Step 2 and the absorbance (C-Abs) obtained in Step 3 is below a preset threshold, the sample contains a carbapenemase-producing bacterium Process to determine
  • a sample for absorbance measurement is prepared. Specifically, it can be prepared by adding an antibacterial agent to the sample and culturing.
  • a sample that can be used since the presence or absence of a carbapenemase-producing bacterium is detected, a sample that may contain the bacterium can be used.
  • biological samples derived from humans and other animals, environmental samples, and food-derived samples are exemplified.
  • biological specimens include clinical materials such as urine, blood, sputum, and stool.
  • environment-derived specimens include water and soil in water and sewage systems, hospital facilities, and medical equipment surfaces (for example, bedside fences, floors, sinks, artificial respirators, etc.).
  • the concentration of the carbapenemase-producing bacterium in the sample is not particularly limited.
  • the sample is added to a medium and cultured under conditions suitable for culturing carbapenemase-producing bacteria, for example, pH 6.0 to 8.0, 10 to 42 ° C.
  • the culture time may be 15 minutes, and from the viewpoint of improving accuracy, the lower limit is exemplified by 30 minutes, 1 hour, 1 hour 30 minutes, and 2 hours.
  • the upper limit 6 hours, 4 hours, and 3 hours are mentioned from a rapid viewpoint.
  • a known medium can be appropriately selected and used.
  • the antibacterial agent to be added is not particularly limited as long as it is an antibacterial agent that can be hydrolyzed by carbapenemase-producing bacteria.
  • carbapenem antibiotics penem antibiotics, specifically, imipenem (IPM), panipenem (PAPM), meropenem (MEPM), biapenem (BIPM), doripenem (DRPM), tevipenem (TBPM), Examples include ertapenem (ETPM) and faropenem (FRPM).
  • the added concentration is a concentration at which hydrolysis can be detected by a method described later when contacted with a carbapenemase-producing bacterium. Specifically, 0.1-100 ⁇ g / mL is exemplified in the culture solution.
  • step 2 the absorption in the ultraviolet region is measured for the culture solution thus obtained, that is, the sample for absorbance measurement.
  • the wavelength to be measured is a wavelength (Anm) that is the maximum absorption maximum wavelength of the antibacterial agent used in Step 1, and at least 40 nm, preferably 42 nm or more, more preferably 45 nm or more, further preferably 50 nm or more from the wavelength.
  • the upper limit is not particularly set, but from the viewpoint of specificity, it is preferably 400 nm or less, more preferably 300 nm or less, still more preferably 200 nm or less, still more preferably 150 nm or less, and even more preferably 100 nm or less. ).
  • Table 1 shows an example of combinations of wavelengths (Anm) and wavelengths (Bnm) that can be set in the present invention for antibacterial drugs.
  • the wavelength (Anm) can be set to 297 nm and the wavelength (Bnm) can be set to 350 nm, but is not limited thereto.
  • the wavelength (Anm) may be set as a maximum wavelength by measuring an antibacterial drug alone dissolved in a culture solution in advance, or may be set from known technical information.
  • the wavelength (Anm) may be set to be slightly different from the maximum wavelength, for example, within the range of the maximum wavelength ⁇ 10 nm, preferably the maximum wavelength ⁇ 5 nm, and the wavelength (Anm) and the wavelength (Bnm) are as described above. It is sufficient that the wavelengths are separated.
  • the absorbance measurement is not particularly limited as long as the absorbance at the above-described wavelength can be measured.
  • the absorbance can be measured using a known spectrophotometer.
  • the absorbance (A-Abs) at the wavelength (Anm) and the absorbance (B-Abs) at the wavelength (Bnm) may be measured separately or simultaneously if they are measured for the same sample.
  • the absorbance since a control solution containing only bacteria as a background is not separately prepared, there is a possibility that the bacteria will grow and the absorbance may fluctuate over time, so there is no time difference when measuring separately. It is preferable to measure at the same time. Specifically, for example, it is preferably within 5 minutes, more preferably within 1 minute.
  • the simultaneous measurement includes a mode in which two wavelengths are measured simultaneously without a time difference, and a mode in which measurement is continuously performed as long as the time difference is such that bacteria do not grow.
  • the sample for absorbance measurement may be appropriately concentrated or diluted depending on the absorbance at the wavelength (Anm).
  • the concentration method and the dilution method are not particularly limited, and examples thereof include a method of concentrating by decompressing at a temperature of room temperature or lower, a method of diluting by adding a culture solution, and the like.
  • step 3 the absorbance (B-Abs) obtained in step 2 is applied to a calibration curve to calculate a blank absorbance (C-Abs).
  • Blank absorbance (C-Abs) is characterized in that it is calculated as a blank absorbance at the wavelength (Anm) described above using a separately prepared calibration curve. That is, in the present invention, since the type and amount of bacteria, the composition of the culture solution, and the like differ depending on the sample for absorbance measurement, the absorbance (A-Abs) is measured where the blank value varies depending on the sample and cannot be set unconditionally.
  • One characteristic is that the blank absorbance (C-Abs) can be set without being affected by the blank value by measuring the absorbance (B-Abs) in the same sample as above and applying it to the calibration curve.
  • a calibration curve is created as follows. First, the amount of antibacterial agent added, the type / amount of bacteria, the type / amount of the culture solution, etc. are prepared in advance, and various culture solutions cultured at different wavelengths (Bnm) are prepared. Measure absorbance. Next, except that no antibacterial drug is added, the sample is prepared under the same conditions as described above, and the absorbance at the wavelength (Anm) is measured for the culture medium cultured under the same conditions, and the correlation can be plotted. .
  • a calibration curve shows a good linear relationship (approximate expression) when it is prepared as absorbance at a wavelength (Bnm) on the X axis and blank absorbance at a wavelength (Anm) on the Y axis. Note that the calibration curve can be recalculated from time to time as the data is additionally updated.
  • step 4 if the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold value, If the sample used in step 1 contains a carbapenemase-producing bacterium, and if it is equal to or exceeds, it is determined that the sample used in step 1 does not contain a carbapenemase-producing bacterium.
  • the difference between absorbance (A-Abs) and absorbance (C-Abs) is compared with a preset threshold value.
  • the threshold is an appropriate cut-off value for identifying whether or not hydrolysis has occurred.
  • Threshold value can be set as follows. For multiple known carbapenemase-producing bacteria and carbapenemase non-producing bacteria, the difference between absorbance (A-Abs) and absorbance (C-Abs) can be determined as described above, and the trend can be determined from these values. it can. For example, the difference in absorbance can be set as 0.2, preferably 0.15, more preferably 0.1. Therefore, if the difference between absorbance (A-Abs) and absorbance (C-Abs) is less than the threshold value of 0.2, preferably 0.15, more preferably less than 0.1, it is determined that the sample contains carbapenemase-producing bacteria. , It can be determined that it is not included when it is equal or above.
  • the presence or absence of bacteria can be confirmed by comparing the difference between absorbance (A-Abs) and absorbance (C-Abs) as it is and the threshold, but from the viewpoint of ease of judgment.
  • the numerical value calculated by applying the relational expression may be used for the determination.
  • the determination can be made by calculating the hydrolysis rate of the antibacterial agent from the absorbance (A-Abs) and the absorbance (C-Abs) using the following formula.
  • Hydrolysis rate (%) 100 ⁇ [(A-Abs) ⁇ (C-Abs)] / [(D-Abs) ⁇ (E-Abs)] ⁇ 100
  • A-Abs Absorbance at the wavelength (Anm) of the culture solution measured in step 2
  • C-Abs Blank absorbance of the culture solution obtained in step 3
  • D-Abs Wavelength of PBS containing antibacterial drug at the same concentration as in step 1
  • E-Abs Absorbance at the wavelength (Anm) of PBS Note that the absorbance (D-Abs) and absorbance (E-Abs) are measured each time the absorbance (A-Abs) is measured.
  • PBS phosphate buffer
  • D-Abs absorbance
  • E-Abs absorbance
  • a buffer solution or the like can be used without particular limitation.
  • the obtained hydrolysis rate is compared with a preset threshold value.
  • the threshold value used here can be set by calculating the hydrolysis rate for a plurality of known carbapenemase-producing bacteria and non-carbapenemase-producing bacteria using the above-described method, and grasping the tendency from these values. For example, a hydrolysis rate of 10%, preferably 15%, more preferably 20% can be set as the threshold value. Therefore, when calculating the hydrolysis rate from the difference between absorbance (A-Abs) and absorbance (C-Abs), the hydrolysis rate is equal to or exceeding 10% of the threshold, preferably 15%, more preferably 20%. Can be determined that the sample contains carbapenemase-producing bacteria, and when the sample is lower, it can be determined that it is not included.
  • the threshold value may be acquired separately when measuring the absorbance of the sample, or may be acquired in advance. Moreover, when comparing with the hydrolysis rate of a sample, the analysis result obtained so far may be additionally updated as needed.
  • the preset threshold value with the absorbance or the hydrolysis rate calculated from the absorbance, it can be determined whether or not the sample contains a carbapenemase-producing bacterium.
  • the bacteria thus detected are not particularly limited as long as they are carbapenemase-producing bacteria, and examples thereof include NDM-type carbapenemase-producing bacteria, KPC-type carbapenemase-producing bacteria, OXA-type carbapenemase-producing bacteria, and IMP-type carbapenemase-producing bacteria.
  • Specific examples include Gram-negative bacteria that produce carbapenemases, including Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Pseudomonas aeruginosa, Vibrio cholera, Acinetobacter spp., Aeromonas spp. .
  • the level of hydrolysis performance of carbapenemase-producing bacteria can also be evaluated in comparison with the threshold value in step 4 in the detection method. Specifically, the smaller the calculated difference in absorbance or the higher the hydrolysis rate, the higher the hydrolysis performance of the carbapenemase-producing bacterium contained in the sample, in other words, it is measured in step 2. It can be evaluated that the lower the absorbance at the wavelength (Anm) of the culture solution, the higher the hydrolysis activity of the carbapenemase-producing bacterium.
  • the present invention can also determine the level of hydrolysis performance of a carbapenemase-producing bacterium from the absorbance at a wavelength serving as an index, and therefore provides a method for determining the hydrolysis performance of a carbapenemase-producing bacterium, including the following steps.
  • Step 1 Step of culturing by adding an antibacterial agent to a sample
  • Step 2 Absorbance (A-Abs) at the maximum wavelength (Anm) of the antibacterial agent with respect to the culture solution obtained in Step 1, and the wavelength Step 3 for measuring absorbance (B-Abs) at a wavelength (Bnm) of at least 40 nm higher wavelength from step 3: Applying the absorbance (B-Abs) obtained in step 2 to a calibration curve, and calculating the blank absorbance (C-Abs) Calculation step 4: When the difference between the absorbance (A-Abs) obtained in step 2 and the absorbance (C-Abs) obtained in step 3 is below a preset threshold, the carbapenemase-producing bacterium contained in the sample To determine that the hydrolysis performance of
  • the section in the detection method of the present invention can be referred to.
  • the bacteria whose hydrolysis performance is determined include those detected by the detection method of the present invention.
  • the detecting device of the present invention includes a spectrophotometric measuring device, a processor, and a computer having a memory under the control of the processor.
  • the memory includes the following steps: Absorbance (A-Abs) at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent and a wavelength at least 40 nm higher than the wavelength of the culture solution cultured with the sample and the antibacterial agent added thereto.
  • the present embodiment includes a spectrophotometric measurement device and a computer system connected to the measurement device.
  • the measurement device is a UV measurement device that measures the absorbance of a culture solution obtained by adding a sample and an antibacterial drug, for example, based on two set wavelengths. Measurements at two wavelengths may be performed simultaneously or separately. The interval in separate cases is not particularly limited, but it is preferable to carry out the steps sequentially.
  • the wavelength is automatically set to the maximum absorption maximum wavelength (Anm) and the wavelength (Bnm) away from the wavelength by a certain wavelength to the higher wavelength side by inputting information on the antibacterial agent used in the computer system described later. It can also be set.
  • the measuring device mix the sample and the antibacterial solution with a pH of 6.0 to 8.0 and 10 to 42 ° C for a period of about 15 minutes (the lower limit is 30 minutes, 1 hour, 1 hour 30 Minutes, 2 hours are exemplified, and the upper limit is exemplified by 6 hours, 4 hours, 3 hours), and then the absorbance can be automatically measured as it is.
  • the type of antibacterial agent, amount used, wavelength setting method, and the like can be referred to the detection method section of the present invention.
  • the sample container may be any container capable of UV measurement, and may be used with reference to the section of the detection kit of the present invention, for example.
  • the obtained absorbance is transmitted to the computer system.
  • the computer system executes a program for determining the presence or absence of carbapenemase-producing bacteria in the sample based on the obtained absorbance.
  • the program for determining the presence or absence of carbapenemase-producing bacteria calculates blank absorbance from the obtained absorbance based on an approximate expression stored in a hard disk or the like, and then calculates the hydrolysis rate.
  • the presence or absence of carbapenemase-producing bacteria in the sample is determined using a preset threshold value described on a hard disk or the like.
  • the determination result can be separately printed on a printer or displayed on a display unit (display).
  • the detection device of the present invention may be configured separately so that it can be connected even if the spectrophotometer and the computer system are integrated. Moreover, the apparatus from the absorbance measurement to the determination may be automated. For example, the apparatus may display the determination result with the addition of the sample.
  • the size of the detection device of the present invention is not particularly limited.
  • the spectrophotometric measurement device and the computer system are integrated, and preferably a small size in which the simultaneous measurement and determination of absorbance at two wavelengths are automated and the result display is automated. It may be a device or a device that can be attached to an existing spectrophotometric apparatus.
  • A-Abs Absorbance at the maximum wavelength (Anm) of the maximum absorption of the antibacterial agent and a wavelength at least 40 nm higher than the wavelength of the culture solution cultured with the sample and the antibacterial agent added thereto.
  • a program for causing a computer to execute a step of determining that a sample contains a carbapenemase-producing bacterium when it falls below a set threshold can also be used by being mounted on a known computer as determination software. It is included as one aspect.
  • the present invention is based on the principle of determining the bacteria contained in the sample based on the fluctuation of the ultraviolet absorption of the antibacterial agent to be used. Therefore, if a compound having a known structure is used. For example, those skilled in the art can fully understand that the present invention can also be applied to detection of ⁇ -lactamase producing bacteria.
  • the screening method of the present invention uses a substance that suppresses the activity of a carbapenemase-producing bacterium as a candidate compound, using a change in absorbance at a specific wavelength as an index, and a sample obtained by allowing a test substance to act on a known carbapenemase-producing bacterium. Compare the absorbance with the absorbance of the sample that does not act, and if the absorbance of the sample that acted the test substance exceeds the absorbance of the sample that did not act, the test substance can be selected as a substance that suppresses the activity of the carbapenemase-producing bacteria it can.
  • the absorbance of a sample that does not act on the test substance is not prepared separately.
  • the absorbance at a wavelength set separately is measured. It is characterized by measuring. Specifically, the absorbance (A-Abs) at the wavelength (Anm) of the sample in which the test substance was allowed to act on the carbapenemase-producing bacterium, and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength.
  • the difference between the absorbance (A-Abs) and the absorbance (C-Abs) is a preset threshold, for example, , 0.1, preferably 0.15, more preferably more than 0.2, the test substance is selected as a substance that suppresses the activity of the carbapenemase-producing bacterium.
  • the maximum wavelength (Anm) is not measured in advance, but is estimated and set from the structure based on the common general knowledge of those skilled in the art, and the wavelength (Bnm) is set with reference to the detection method of the present invention. Good.
  • the blank absorbance (C-Abs) is calculated with reference to the detection method section of the present invention. be able to.
  • the carbapenemase-producing bacterium that can be used in the screening method of the present invention is not particularly limited as long as it can be detected by the detection method of the present invention. Culture conditions and the like can be set as appropriate based on the bacteria used.
  • the hydrolysis rate can be calculated and compared by applying the formula of the hydrolysis rate used in the detection method of the present invention.
  • the test substance can be selected as a substance that suppresses the activity of the carbapenemase-producing bacteria, and the hydrolysis rate is 20
  • a test substance exceeding% cannot be selected as a substance that suppresses the activity of a carbapenemase-producing bacterium.
  • the kit for detecting carbapenemase-producing bacteria Since the present invention determines the presence or absence of bacteria based on the change in ultraviolet absorption of the added antibacterial agent, it can be determined by adding an antibacterial agent. It can also be said. Therefore, the present invention also provides a kit for detecting a carbapenemase-producing bacterium containing an antibacterial drug.
  • the kit of the present invention is preferably a kit that can be provided for absorbance measurement by adding a sample to a container.
  • a container plate type, card type, test tube type, etc.
  • the container may be provided with a well, or an antibacterial drug may be dried and fixed on the side surface and / or the bottom surface of the container or each well.
  • a solvent for dissolving the antibacterial drug for example, PBS
  • Such a kit includes, for example, containers for positive control, negative control, and sample preparation. These containers come into direct contact with bacteria, and are preferably disposable in consideration of safety. Moreover, it is preferable that it is applicable to the detection apparatus of this invention.
  • an antibacterial agent added in the detection method of the present invention can be used.
  • the concentration of the antibacterial agent in the kit is not particularly limited as long as the concentration of the antibacterial agent in the culture solution is, for example, 0.1 to 100 ⁇ g / mL at the time of measuring ultraviolet absorption, and can be set as appropriate.
  • materials used for sample preparation for absorbance measurement can also be included.
  • a known medium or the like can be applied as long as the bacteria to be measured can be cultured.
  • imipenem was used from Wako Pure Chemical Industries, Ltd .
  • Meropenem was Tokyo Chemical Industry Co., Ltd .
  • Doripenem, Biapenem, Eltapenem, Faropenem and PBS (phosphate buffer) were purchased from Sigma-Aldrich.
  • Test example 1 Prepare UV solution 96-well plate (Zell-tive, Germany) in PBS solution with various concentrations of each antibacterial agent, using corona plate reader SH-9000 (HITACHI, Japan), room temperature (25 ° C) Below, analysis was performed at wavelengths between 220 nm and 400 nm (FIG. 1 a).
  • Each antibacterial solution showed a characteristic absorbance between 250 nm and 350 nm, and peak absorbance around 297 nm. Further, even if the spectrum was analyzed up to 1000 nm, no other peak was identified.
  • the absorbance at 297 nm (A-Abs) showed a linear relationship with the concentration in the solution (FIG. 1b), and the abundance in the solution could be estimated from the absorbance of imipenem.
  • Test example 2 The spectrum of imipenem in the bacterial mixture was measured to see if a difference in hydrolysis activity could be found between carbapenemase-producing bacteria (CPE strain) and carbapenemase non-producing bacteria (non-CPE strain) .
  • CPE strains Klebsiella pneumoniae, ATCC BAA-2470 strain
  • non-CPE strains Klebsiella pneumoniae, ATCC 4352 strain
  • CPE imipenem and CPE strain
  • PBS PBS
  • a mixture of the antibacterial agent and the CPE strain and a suspension of the CPE strain in PBS were prepared, and the spectrum from 200 nm to 1000 nm was also measured.
  • both antibacterial agents are a mixture of antibacterial agent and CPE strain (antibacterial agent + Bac) and a suspension of CPE strain in PBS (PBS + Bac) shows that the spectra are almost identical.
  • Test example 3 Since the background seemed to change due to different types of bacteria, "absolute absorbance of imipenem” in the bacterial mixture was examined. As shown in FIG. 3a, for example, at a wavelength of 297 nm, the absolute absorbance of imipenem is obtained by subtracting the absorbance (point C) of the bacterial mixture containing no imipenem from the absorbance (point A) of the bacterial mixture containing imipenem. Therefore, the absolute absorbance of the sample of Test Example 2 was calculated as described above.
  • FIG. 3b clearly shows that the hydrolysis of imipenem is clearly shown as the disappearance of the characteristic spectrum of imipenem in the solution containing CPE, and the presence or absence of CPE can be detected by the difference in absorbance.
  • the non-CPE solution for example, had a relatively stable peak absorbance at 297 nm and was similar to the imipenem solution without bacteria.
  • Test example 4 In the method of Test Example 3, it was necessary to prepare two types of solutions for measuring the absorbance. Specifically, a solution containing bacteria and imipenem (experimental solution) and a solution containing only bacteria at the same concentration (background) were required (FIG. 4a). However, considering application to the detection system, it was ideal to skip the procedure of preparing the background solution in order to save time and simplify the system. Also, the extent of bacterial growth in the two solutions was different, suggesting that the background solution could no longer be used as “background”. Therefore, as shown in FIG. 4b, a method for estimating background absorbance using an experimental solution was examined.
  • Test Example 5 Based on Test Example 4, the hydrolysis activity of 55 known strains shown in Table 3 was measured. The control strains purchased from ATCC were used, and the others were human clinical isolates owned by the laboratory.
  • the bacteria are cultured overnight on an agar plate, and the colony is added to 100 ⁇ L of imipenem solution (25 ⁇ g / mL) to achieve a turbidity equivalent to 1.0 McFarland standard, then at 37 ° C. for 60 minutes. After the incubation, spectrophotometric measurements were taken. From the obtained absorbance, the hydrolysis rate was calculated using the following formula.
  • Hydrolysis rate (%) 100 ⁇ [(A-Abs) ⁇ (C-Abs)] / [(D-Abs) ⁇ (E-Abs)] ⁇ 100
  • E-Abs PBS Absorbance at wavelength (297nm)
  • Fig. 6a shows that the hydrolysis rate is clearly separated between CPE and non-CPE strains.
  • the MIC value of imipenem is shown using color scale, some CPEs have low MIC values, but the difference in carbapenemase activity from non-CPE strains can be achieved by using the detection method of the present invention. Is clear.
  • OXA type which is known to have a weak hydrolysis action in a short time, has been confirmed to exhibit relatively lower activity than other CPE strains, and the detection method of the present invention is also suitable for such detection. It was found to be excellent (Figure 6b).
  • Test Example 6 About the carbapenemase producing bacteria (CPE strain: Klebsiella pneumoniae, ATCC BAA-2470 strain) and the carbapenemase non-producing bacteria (non-CPE strain: Klebsiella pneumoniae, ATCC 4352 strain) used in Test Example 5 using each antibacterial agent, Test Example 5 The hydrolysis rate was calculated in the same manner as above. In this test, the approximate expression for calculating the blank absorbance was that of imipenem prepared in Test Example 4, and for each antibacterial agent, the wavelength (Anm) was set to 297 nm and the wavelength (Bnm) was set to 350 nm for convenience.
  • CPE strain Klebsiella pneumoniae, ATCC BAA-2470 strain
  • non-CPE strain Klebsiella pneumoniae, ATCC 4352 strain
  • Test Example 7 It was confirmed whether the detection method of the present invention can cope with various strains. Specifically, hydrolytic activity was evaluated for the strains shown in Table 4 (149 strains) in the same manner as in Test Example 4 except that the incubation time was changed to 120 minutes. The strain was purchased from ATCC or a human clinical isolate owned by the laboratory.
  • the detection method of the present invention is an excellent detection method applicable to various strains.
  • Test Example 8 Regarding the detection method of the present invention, the discrimination between CPE strains and non-CPE strains at different incubation times (30, 60, 120, 180 minutes) was examined using 149 strains used in Test Example 7. Specifically, the hydrolysis activity was evaluated in the same manner as in Test Example 4 except that the incubation time was changed to the above time. Further, at each incubation time, an ROC curve was created regarding the hydrolysis rate, and a cut-off value was calculated (Table 5).
  • the cut-off value 9.43 after incubation for 30 minutes has a sensitivity of 98.25% and a specificity of 100%, indicating that the judgment performance is sufficient.
  • the incubation time is 60 minutes, 120 minutes, and 180 minutes, 100% sensitivity and specificity are exhibited, and it can be seen that the detection method of the present invention can perform determination with good specificity and accuracy (FIG. 9). ).
  • Test Example 9 An antibacterial agent is added to a colony grown by culturing a specimen (blood, etc.) derived from a biological sample and further cultured. For the obtained culture solution, the absorbance (A-Abs) at the maximum wavelength (Anm) and the absorbance (B-Abs) at a wavelength (Bnm) at least 40 nm higher than the wavelength are measured according to the added antibacterial agent. The blank absorbance (C-Abs) is calculated from the absorbance (B-Abs). Next, it is determined from the difference between the absorbance (A-Abs) and the blank absorbance (C-Abs) whether the specimen contains a carbapenemase-producing bacterium.
  • the detection method of the present invention can detect carbapenemase-producing bacteria easily and rapidly with high sensitivity and specificity, and is useful for clinical and hospital infection countermeasures.

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Abstract

L'invention concerne un procédé de détection d'une bactérie productrice de carbapénémase, comprenant : une étape 1 qui est une étape d'addition d'un médicament antibactérien à un échantillon et de culture de l'échantillon ; une étape 2 qui est une étape de mesure d'une absorbance (A-Abs) d'une culture obtenue à l'étape 1 à une longueur d'onde maximale (Anm) d'une absorption maximale du médicament antibactérien et d'une absorbance (B-Abs) de la culture à une longueur d'onde (Bnm) qui est plus longue d'au moins 40 nm que la longueur d'onde (Anm) ; une étape 3 qui est une étape d'attribution de l'absorbance (B-Abs) obtenue à l'étape 2 à une courbe d'étalonnage pour calculer une absorbance à blanc (C-Abs) ; et une étape 4 qui est une étape de détermination du fait qu'une bactérie productrice de carbapénémase est contenue dans l'échantillon lorsque la différence entre l'absorbance (A-Abs) obtenue à l'étape 2 et l'absorbance (C-Abs) obtenue à l'étape 3 est inférieure à une valeur seuil prédéterminée. Selon le procédé de détection de la présente invention, une bactérie productrice de carbapénémase peut être détectée de manière commode et rapide et avec une sensibilité élevée et une spécificité élevée. Par conséquent, le procédé de détection est utile dans des pratiques cliniques et en tant que mesure contre une infection acquise à l'hôpital.
PCT/JP2018/013920 2017-03-31 2018-03-30 Procédé pour la détection d'une bactérie produisant de la carbapénémase WO2018181997A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015191907A1 (fr) * 2014-06-11 2015-12-17 VenatoRx Pharmaceuticals, Inc. Inhibiteurs de bêta-lactamases
WO2016156605A1 (fr) * 2015-04-03 2016-10-06 Universite De Bourgogne Nouvelle methode pour detecter la presence de bacteries productrices de beta-lactamases
JP2016182066A (ja) * 2015-03-26 2016-10-20 栄研化学株式会社 β−ラクタマーゼの検出法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015191907A1 (fr) * 2014-06-11 2015-12-17 VenatoRx Pharmaceuticals, Inc. Inhibiteurs de bêta-lactamases
JP2016182066A (ja) * 2015-03-26 2016-10-20 栄研化学株式会社 β−ラクタマーゼの検出法
WO2016156605A1 (fr) * 2015-04-03 2016-10-06 Universite De Bourgogne Nouvelle methode pour detecter la presence de bacteries productrices de beta-lactamases

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